Yersinia enterocolitica has been implicated as a significant cause of enteritis and enterocolitis, may cause mesenteric adenitis, hepatosplenic abcesses, and septicemia. Virulence of Y. enterocolitica is associated with a 42-46Md plasmid (pYV). While a number of "virulence" characteristics (calcium dependence, expression of specific outer membrane proteins, autoagglutination, uptake of Congo red or crystal violet, etc.) are known to be encoded by pYV, we still have only a limited understanding of how these and other plasmid factors contribute to pathogenicity. To address this question we subcloned the BamHI and SalI restriction fragments of pYV from Y. enterocolitica strain A2635 into pRK290 derivatives, and introduced these subclones into plasmid- cured Y. enterocolitica host strains. We have subsequently identified plasmid gene sequences that influence calcium dependence, uptake of crystal violet, expression of the Pl plasmid-associated outer membrane protein, and cytotoxicity for HEp-2 cells. We have also identified a 2.8kb BamHI fragment that encodes factors responsible for conjunctivitis in guinea pigs (the Sereny reaction). This proposal will continue these studies, with an emphasis on identification and characterization of plasmid factors responsible for virulence in animal models. We will sequence our 2.8kb conjunctivitis- associated fragment, and will further characterize protein(s) produced by this and other plasmid fragments. We will then mutagenize gene sequences encoding selected plasmid factors, and introduce these mutations into an intact virulence plasmid by marker exchange; host strains containing specific mutations in the virulence plasmid will be screened in vitro and in animal models in an effort to define the role of each factor in pathogenicity. Finally, by introducing combinations of "critical" plasmid gene sequences back into Y. enterocolitica, we will try to determine the combination of plasmid genes sufficient for full expression of virulence. These data should provide us with a basic understanding of plasmid-mediated factors and their role in the pathogenesis of Y. enterocolitica infections. Our results have practical implications for development of screening assays for virulent strains, and potential applications in vaccine development; results should also contribute to our overall understanding of the pathogenesis of enteric infections.